Establishment of a pheasant (Phasianus colchicus) spermatogonial stem cell line for the production of interspecies germ line chimeras

Abstract

a b s t r a c tBackground: Spermatogonial stem cells (SSCs) are important for the production of interspecies germ line chimeras. The interspecies germ cell transfer technique has been suggested as a way to conserve endangered birds. Our objective was to develop a technique for restoring endangered birds by developing interspecies germ line chimeras between pheasant (Phasianus colchicus) and chicken (Gallus gallus) with SSCs. Results: SSCs were isolated from the surgically removed testis of a pheasant. Growth conditions for pheasant SSCs were established by co-culturing STO (SIM mouse embryo-derived thioguanine and ouabain resistant) cells and pheasant SSCs. The colony-forming cells divided and proliferated stably to yield an established SSC line. Pheasant SSCs showed strong reactivity for GDNF family receptor alpha1 (GFRα1) marker. Finally, production of germ line chimeras was attempted by transferring pheasant SSCs into recipient embryos. Although final embryo survival was 5.6% (20/354), the initial survival rate was 88% (312/354). To measure the percent transfer of donor SSC to gonads, the pheasant SSCs were labeled with PKH 26 fluorescent dye. We observed 30% donor cells and 9.48% c-kit/CD117-positive cells in the gonads of recipient chickens. Donor SSCs were thus stably engrafted in the recipient gonads.Conclusions: This study showed that SSCs can be used as a tool for the conservation of endangered birds and the production of germ line chimeras. Our findings yield insights into how we may use the pheasant spermatogonial stem cell line for efficient production of interspecies germ line chimeras and ultimately, to the restoration of endangered birds.

Models and Molecular Markers of Spermatogonial Stem Cells in Vertebrates: To Find Models in Nonmammals

“…GFR α -1 mRNA and protein expression is detected mainly in type A SPG close to the basement membrane of the seminiferous tubule, and GFR α -1-expressing SPG produce the progenies in recipient chickens [ 95 , 96 ]. Spermatogonia, isolated from juvenile and adult quail using a differential plating technique, express GFR α -1, and SPG cultured from adult pheasant testes also express GFR α -1 [ 97 , 98 ]. Mammalian GFR α -1 is expressed in gonocytes and undifferentiated type A SPG of the testis and cultured (or isolated) SSCs (Supplementary Table 1 ).…”

Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

“…SSCs, the other type of male germline-competent stem cells in adult testes that are precursors in spermatogenesis, have been successfully isolated from mammalian species, cultured, and used for transgenic production 14 , 15 . SSCs of several bird species including chickens, pheasants, and quails have been isolated and cultured, but practical applications for producing transgenic birds using these cells are limited 16 – 20 . In zebra finch, although studies have reported the isolation and manipulation of PGCs, reports of exogenous genes being efficiently delivered into these cells are limited 21 , 22 .…”